Chromatin was prepared from HEK-293T transfected with myc-His tagged Histone H3.3 mutated G34V and Histone H3.3 WT cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with then formaldehyde for 10min.
The ChIP was performed with 25 µg of chromatin, 2 µg of ab254401 (red), or 2 µg of rabbit normal IgG ab172730 (gray) and 20 µl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).
Primers and probes are located in the first kb of the transcribed region.

*https://www.abcam.com/resources?keywords=X%20ChIP%20protocol

All lanes : Anti-Histone H3.3 (mutated G34V) antibody [EPR23520-5] - ChIP Grade (ab254401) at 1/1000 dilution

Lane 1 : HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34V expression vector containing a myc-His-tag®, whole cell lysate
Lane 2 : HEK-293T transfected with Histone H3.3 (WT) expression vector containing a myc-His-tag®, whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 15 kDa
Observed band size: 20 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

Exposure time: 10 seconds.

Histone H3.3(mutated G34 V) was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34V expression vector containing a myc-His-tag^® whole cell lysate 10 µg with ab254401 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254401 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HEK-293T transfected with Histone H3.3 G34V expression vector containing a myc-His-tag^® whole cell lysate 10 µg

Lane 2: ab254401 IP in HEK-293T transfected with Histone H3.3 G34V expression vector containing a myc-His-tag^® whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254401 in HEK-293T transfected with Histone H3.3 G34V expression vector containing a myc-His-tag^® whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 32 seconds.

 

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HEK-293T (Human embryonic kidney epithelial cell) transfected with myc tagged Histone H3.3 WT construct (Left panel) and myc-tagged Histone H3.3 H3G34V construct (Right panel) cells labelling Histone H3.3(mutated G34 V) with ab254401 at 1/500 dilution (0.1µg). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T cells labelling Histone H3.3(mutated G34 V) with ab254401 at 1/1000 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 2 µg/ml dilution (Green). Confocal image showing nuclear staining in HEK-293T cells transfected with Histone H3.3 H3G34V-Myc plasmid, while no staining in HEK-293T cells transfected with H3.3 WT -Myc plasmid. Myc-Tag Mouse mAb (Alexa Fluor^® 647) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 2 µg/ml dilution.

Immunohistochemical analysis of paraffin-embedded human giant cell tumor of bone tissue labeling Histone H3.3(mutated G34 V) with ab254401 at 1/1000 dilution (0.542 µg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human giant cell tumor of bone. (PMID: 29241742). The section was incubated with ab254401 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human chondroblastoma tissue labeling Histone H3.3(mutated G34 V) with ab254401 at 1/1000 dilution (0.542 µg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Negative control: No staining on human chondroblastoma (PMID: 29241742).

The section was incubated with ab254401 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Dot blot analysis of Histone H3.3 (mutated G34 V) labeled with ab254401 at 1/1000 dilution.

Lane 1: Histone H3.3 H3G34V peptide (aa28-40).
Lane 2: Histone H3.3 H3G34V peptide (aa26-38).
Lane 3: Histone H3.3 WT peptide (aa26-40).

Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution was used as secondary antibody.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.